Comparing common and rare single variant and gene aggregate instrumentation strategies for MR

Introduction

Classically, Mendelian Randomisation (MR) methods utilising trait-associated genetic variants from GWAS studies have employed common polymorphisms (population MAF > 1%) targeted by genotyping chips, or reliably imputed from reference populations, to instrument modifiable exposures. However, common variants tested in GWAS typically explain a very small fraction of the variability in a measured complex trait, potentially exhibit pleiotropic effects acted upon by balancing selection or as a consequent of genetic linkage, and are rarely causal. Rare variants, which typically show large biological effects (e.g. through abolishing protein expression) provide a means of more unambiguously instrumenting relevant molecular processes. Comparison of causal estimates derived using differing methods of genetically instrumenting modifiable exposures may enhance the interpretation of the biological mechanisms underlying exposure-outcome relationships. This includes using variants from across the allele frequency spectrum, but also leveraging rare variant aggregate approaches to instrument gene-level perturbations in expression and function.

Causal estimates for pairwise combinations of the exposure and outcome relationships below have been derived using twelve instrumentation strategies.

Exposures: Low density lipoprotein levels (LDL direct), Body Mass Index (BMI), Vitamin D, Triglycerides, Glycated Haemoglobin (HbA1c), Mean Platelet Volume (MPV), IGF-1, Waist-to-Hip Ratio (BMI-corrected), Red Blood Cell (RBC) erythrocyte count and Mean Corpuscular Volume (MCV)

Outcomes: Coronary Artery Disease (CAD), Type 2 Diabetes (T2D), Multiple Sclerosis (MS), Ischemic Stroke, Atrial Fibrillation (AF), Venous Thromboembolism (VTE), Prostate Cancer and Hypertension.

Instruments

Twelve sets of instruments for each exposure have been extracted from across three sources (DeepRVAT gene impairment scores, Genebass whole exome single variants and aggregate burden masks and UKB common variant GWAS summary statistics):

Common GWAS
Associated common variants from UKB GWAS (>1% MAF)

Genebass (variants)
Common exome-wide (>5% MAF, LD clumped)
Low-frequency exome-wide (1-5% MAF, LD clumped)
Rare exome-wide (0-1% MAF, both unfiltered and filtered to the top hit per gene)
Ultra-rare exome-wide (0-0.1% MAF, both unfiltered and filtered to the top hit per gene)

Genebass (masks)
pLOF burden mask
missense/low-confidence burden mask
pLOF/missense/low-confidence burden mask
synonymous burden mask

DeepRVAT (impairment scores)
gene impairment score (<0.1% MAF)

Here the focus is on 10 of the tested exposure-outcome pairs where differing instrumentation strategies show heterogeneity in causal effect estimates.

Data import

Read in MR results from scripts/02_analysis_complextraits/001_performMR.R and harmonised summary statistics:

# Import MR results
load(file = file.path(res_dir, "results_complextrait_MR.rda"))
# Import harmonised study data
load(file = file.path(data_dir, "harmonised", "harmonised_studies.rda"))

# Retain exposure-outcome pairs with significant IVW estimate for at least one instrumentation strategy
res_MR <- lapply(results_MR, function(x){
  sig_ivw <- x |> filter(method == "Inverse variance weighted" & pval < 0.05)
  if(nrow(sig_ivw) == 0){
    return(NULL)
  } else {
    return(x)
  }
}) 

keep_pairs <- names(unlist(lapply(res_MR, nrow)))
# Remove BMI exposure pairs (low instrument numbers)
keep_pairs <- keep_pairs[!(grepl("^BMI",keep_pairs))] # 44 exposure-outcome pairs

# Top interesting
keep_top <- c(
  "HbA1c_CHD", "HbA1c_T2D", "LDL_CHD", "LDL_MS", "LDL_T2D", "Trig_CHD", 
  "Trig_T2D", "WHRadjBMI_CHD", "WHRadjBMI_Hypertension", "WHRadjBMI_T2D")

res_MR <- res_MR[keep_top]

# Annotations for study sets
instrument_type <- c("genescore",rep("mask",4), rep("variant",7))
names(instrument_type) <- names(harmonised_studies[[1]])

instrument_class <- c("genescore",
                      "mask:missense|LC","mask:pLOF","mask:pLOF|missense|LC","mask:synonymous",
                      "exome:common","exome:lowfreq","exome:rare","exome:ultrarare",
                      "exome:rare (top)","exome:ultrarare (top)","genome:common")

names(instrument_class) <- names(harmonised_studies[[1]])

# Populate missing MAFs from other paired studies if possible

all_mafs <- list()

for(i in 1:length(harmonised_studies)){
  snp_mafs <- do.call("rbind",lapply(harmonised_studies[[i]][names(instrument_type[instrument_type=="variant"])], function(x){
    x[,c("SNP","effect_allele.exposure","eaf.exposure")]
  })) |> unique()
  all_mafs[[i]] <- snp_mafs
}

all_mafs <- do.call("rbind",all_mafs) |> arrange(SNP) |> filter(!is.na(eaf.exposure), duplicated(SNP) == FALSE)

for(i in 1:length(harmonised_studies)){
  for(j in 1:length(harmonised_studies[[i]])){
    
    snp_id <- harmonised_studies[[i]][[j]]$SNP
    if (all(is.na(harmonised_studies[[i]][[j]]$eaf.exposure))){
      harmonised_studies[[i]][[j]]$eaf.exposure <- all_mafs[match(snp_id, all_mafs$SNP),"eaf.exposure"]
    }
    if (all(is.na(harmonised_studies[[i]][[j]]$eaf.outcome))){
      harmonised_studies[[i]][[j]]$eaf.outcome <- all_mafs[match(snp_id, all_mafs$SNP),"eaf.exposure"]
    }
  }
}

harmonised_studies <- harmonised_studies[keep_top]

# Add columns with trait pairing and instrument class
for(i in 1:length(harmonised_studies)){
  for(j in 1:length(harmonised_studies[[i]])){
    harmonised_studies[[i]][j][[1]]$pair <- names(harmonised_studies)[i]
    harmonised_studies[[i]][j][[1]]$class <- as.character(instrument_class[names(harmonised_studies[[i]][j])])
  }
}

Add gene annotations for instruments (taken from variant/mask position in exome data for ExWAS studies and nearest gene for common variant GWAS studies)

# Annotate DeepRVAT and burden masks with relevant gene
# ExWAS single variants are already annotated
# Annotate common variants with nearest gene based on VEP annotation:

# List of rsIDs to extract from VEP file (using VEP online interface, returning single consequence per variant --pick)
# common_instruments <- unlist(lapply(harmonised_studies, function(x){
#   return(x$opengwas_common$SNP)
# })) |> unique()

# write.table(common_instruments, file.path(data_dir,"variant_annotation","complextrait_openGWASinstruments.txt"), 
#             row.names = F, col.names = F, quote = F)

vep <- data.table::fread(file.path(data_dir,"variant_annotation","vep_openGWASinstruments.txt")) |>
  dplyr::filter(grepl("^[0-9]",Location))

## Retain variant annotations for SNPs within 1kb of a protein coding gene only
vep_coding <- vep |> 
  filter(BIOTYPE == "protein_coding") |>
  filter(!(as.numeric(DISTANCE) > 1000) | DISTANCE == "-")

for(i in 1:length(harmonised_studies)){
  for(j in 1:length(harmonised_studies[[i]])){
    
    if(names(harmonised_studies[[i]][j]) == "deeprvat_genescore"){
      harmonised_studies[[i]][[j]]$gene.exposure = toupper(harmonised_studies[[i]][[j]]$SNP)
    }
    
    else if(names(harmonised_studies[[i]][j]) %in% names(instrument_type[instrument_type == "mask"])){
      harmonised_studies[[i]][[j]]$gene.exposure = toupper(gsub("_.*","",harmonised_studies[[i]][[j]]$SNP))
    }
    
    else if (names(harmonised_studies[[i]][j]) == "opengwas_common"){
      gene_symbols <- vep_coding[match(harmonised_studies[[i]][[j]]$SNP, vep_coding$`#Uploaded_variation`),c("SYMBOL","Consequence")] |> as.data.frame()
      names(gene_symbols) <- c("gene.exposure","vep.consequence")
      
      harmonised_studies[[i]][[j]] <- cbind(harmonised_studies[[i]][[j]], gene_symbols)
    }
    
    else{
      next
    }
  }
}

Check instrument selection

Single variant instruments only:

# Check number of instruments, minor allele frequency and average beta values across instrument sets
instrument_MAFs <- list()

for(i in 1:length(harmonised_studies)){
  MAFs <- do.call("rbind", lapply(harmonised_studies[[i]], function(x){
    x |> 
      mutate(maf = ifelse(eaf.exposure > 0.5, 1-eaf.exposure, eaf.exposure)) |>
      group_by(exposure,outcome) |>
      dplyr::summarise(
        n_instruments = n(),
        mean_maf = mean(na.exclude(maf)),
        mean_beta = mean(abs(beta.exposure)))
  }))
  MAFs$pairs <- names(harmonised_studies)[i]
  MAFs$study <- names(harmonised_studies[[i]])
  MAFs$instrument_type <- instrument_type[MAFs$study]
  MAFs$instrument_class <- instrument_class[MAFs$study]
  
  instrument_MAFs <- c(instrument_MAFs, list(MAFs))
}

instrument_MAFs <- do.call("rbind", instrument_MAFs) |>
  mutate(instrument_class = factor(instrument_class, levels = c(
    "genome:common",
    "exome:common","exome:lowfreq",
    "exome:rare","exome:rare (top)",
    "exome:ultrarare","exome:ultrarare (top)",
    "mask:synonymous","mask:missense|LC",
    "mask:pLOF|missense|LC","mask:pLOF",
    "genescore")))

## Plot average instrument MAF

p_MAF <- ggplot(instrument_MAFs |> filter(instrument_type == "variant"),
       aes(x = instrument_class, y = mean_maf, size = n_instruments)) +
  geom_point() + 
  facet_wrap(.~pairs, nrow = 2) +
  geom_hline(yintercept = 0.01, lty = 2) +
  geom_hline(yintercept = 0.05, lty = 2) +
  geom_hline(yintercept = 0.001, lty = 2) +
  scale_y_log10(labels = scales::comma, limits = c(0.0001,1)) +
  theme_bw() +
  theme(legend.position = "bottom",
        axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.5),
        strip.text = element_text(size = 6.5)) +
  labs(y = "Mean instrument MAF", x = "Source study", 
       size = "n (instruments)")

p_MAF

## Plot average instrument beta

p_beta <- ggplot(instrument_MAFs |> filter(instrument_type == "variant"),
       aes(x = instrument_class, y = mean_beta, size = n_instruments)) +
  geom_point() + 
  facet_wrap(.~pairs, nrow = 2) +
  theme_bw() +
  theme(legend.position = "bottom",
        axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.5),
        strip.text = element_text(size = 6.5)) +
  labs(y = "Mean instrument |exposure beta|", x = "Source study", 
       size = "n (instruments)")

p_beta

Instrument gene coverage

Are the genes being hit by common and rare variant derived instrument sets for complex exposures overlapping?

# Extract the maximal set of gene annotations for each exposure instrument set

instrument_genes <- do.call("rbind", lapply(harmonised_studies, function(x){
  do.call("rbind", lapply(x, function(y){
    y[,c("pair","class","gene.exposure")]
  }))
})) |> 
  mutate(exposure = sub("_.*","",pair),
         hit = 1,
         class = factor(class, 
                        levels = c(
                          "genome:common",
                          "exome:common","exome:lowfreq",
                          "exome:rare","exome:rare (top)",
                          "exome:ultrarare","exome:ultrarare (top)",
                          "mask:synonymous","mask:missense|LC",
                          "mask:pLOF|missense|LC","mask:pLOF",
                          "genescore"))) |>
  filter(!(class %in% c("exome:rare (top)", "exome:ultrarare (top)"))) |>
  select(-pair) |> unique()
rownames(instrument_genes) <- NULL

# Is gene hit by at least one common, rare (or ultra rare) and mask (or genescore)
instrument_genes <- instrument_genes |>
  mutate(set = ifelse(class %in% c("genome:common","exome:common","exome:lowfreq"), "common", 
                      ifelse(class %in% c("exome:rare","exome:ultrarare"), "rare",
                             ifelse(grepl("mask|genescore", class), "aggregate", NA))),
         set_variants = ifelse(class %in% c("genome:common","exome:common","exome:lowfreq"), "common", 
                      ifelse(class %in% c("exome:rare","exome:ultrarare"), "rare", NA)))

n_sets <- instrument_genes |> group_by(gene.exposure, exposure, set) |> summarise(count = n()) |> 
  group_by(gene.exposure,exposure) |> summarise(count_sets = n())
n_sets_variants <- instrument_genes |> na.exclude() |> group_by(gene.exposure, exposure, set_variants) |> summarise(count = n()) |> 
  group_by(gene.exposure,exposure) |> summarise(count_sets_variants = n())

instrument_genes <- left_join(instrument_genes, n_sets, by = c("gene.exposure","exposure")) |>
  left_join(n_sets_variants, by = c("gene.exposure","exposure")) |>
  mutate(colour_sets = ifelse(count_sets == 3,1,
                              ifelse(count_sets < 3 & count_sets_variants == 2,2,3)))
instrument_genes$colour_sets[is.na(instrument_genes$count_sets_variants)] <- 3

p_genes <- ggplot(instrument_genes, aes(y = gene.exposure, x = class)) +
  facet_wrap(exposure ~ ., nrow = 1, scale = "free_y") +
  geom_tile(aes(fill = as.factor(colour_sets))) +
  scale_fill_manual(values = c("tomato", "orange","darkblue")) +
  theme_bw() +
  theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.5),
        axis.text.y = element_text(size = 5),
        legend.position = "none",
        plot.title = element_text(hjust = 0.5)) +
  labs(title = "Gene containing (or proximal to) instruments for exposures", y = "", x = "Instrument class")
        
p_genes

Mendelian randomisation

IVW estimates across instrument sets

res_IVW <- do.call("rbind", res_MR) |> filter(method == "Inverse variance weighted") |>
  mutate(instrument_type = instrument_type[study],
         instrument_class = factor(instrument_class[study], 
                                   levels = c(
                                     "genome:common",
                                     "exome:common","exome:lowfreq",
                                     "exome:rare","exome:rare (top)",
                                     "exome:ultrarare","exome:ultrarare (top)",
                                     "mask:synonymous","mask:missense|LC",
                                     "mask:pLOF|missense|LC","mask:pLOF",
                                     "genescore")))

ggplot(res_IVW, aes(x = b, y = instrument_class, colour = instrument_type)) +
  facet_wrap(.~pair, nrow = 2, scale = "free_x") +
  geom_vline(xintercept = 0, colour = "grey20", lty = 2) +
  geom_point(aes(pch = pval<0.05, size = nsnp)) +
  geom_errorbar(aes(xmin = b-1.96*se, xmax = b+1.96*se), width = 0.1) +
  scale_colour_manual(values = c("tomato","orange","darkblue")) +
  scale_shape_manual(values = c(1,19)) +
  theme_bw() +
  labs(size = "n (instruments)", colour = "Type", x = "IVW", y = "")

IVW estimates across instrument sets (subsetting to shared genes)

Difference in causal effect estimates across instrument sets could be due to differences in the underlying biological processes that are being captured by the included variants. The above analysis was repeated with restriction to instruments hitting a common set of genes/gene regions across strategies:

# Keep genes in three or more of the instrument sets: genome:common, exome:common, exome:rare, mask:pLOF|missense|LC
shared_genes <- instrument_genes |> filter(class %in% c("genome:common","exome:common","exome:rare","mask:pLOF|missense|LC")) |>
  group_by(gene.exposure, exposure) |> mutate(count = n()) |> filter(count >= 3) |> ungroup()

p_genes_shared <- ggplot(shared_genes, 
       aes(y = gene.exposure, x = class)) +
  facet_wrap(exposure ~ ., nrow = 1, scale = "free_y") +
  geom_tile(fill = "tomato") +
  theme_bw() +
  theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.5),
        axis.text.y = element_text(size = 5),
        legend.position = "none",
        plot.title = element_text(hjust = 0.5)) +
  labs(title = "Gene containing (or proximal to) instruments for exposures", y = "", x = "Instrument class")
        
harmonised_studies_sharedgenes <- harmonised_studies[grepl("HbA1c|LDL|Trig", names(harmonised_studies))] # Subset exposures
harmonised_studies_sharedgenes <- lapply(harmonised_studies_sharedgenes, function(x){
  x[which(names(x) %in% c("opengwas_common", "genebass_common", "genebass_rare","genebass_pLoF|missense|LC"))]}) # Subset instrument sets

# Subset shared genes from instrument sets for each exposure
harmonised_studies_sharedgenes[c("HbA1c_CHD","HbA1c_T2D")] <- lapply(harmonised_studies_sharedgenes[c("HbA1c_CHD","HbA1c_T2D")], function(x){
  lapply(x, function(y){y |> filter(gene.exposure %in% (shared_genes |> filter(exposure == "HbA1c") |> pull(gene.exposure)))})
})
harmonised_studies_sharedgenes[c("LDL_CHD","LDL_MS","LDL_T2D")] <- lapply(harmonised_studies_sharedgenes[c("LDL_CHD","LDL_MS","LDL_T2D")], function(x){
  lapply(x, function(y){y |> filter(gene.exposure %in% (shared_genes |> filter(exposure == "LDL") |> pull(gene.exposure)))})
})
harmonised_studies_sharedgenes[c("Trig_CHD","Trig_T2D" )] <- lapply(harmonised_studies_sharedgenes[c("Trig_CHD","Trig_T2D" )], function(x){
  lapply(x, function(y){y |> filter(gene.exposure %in% (shared_genes |> filter(exposure == "Trig") |> pull(gene.exposure)))})
})

# Run MR on restricted instrument set
results_MR_sharedgenes <- list()
results_MR_sharedgenes_SNP <- list()

for(i in 1:length(harmonised_studies_sharedgenes)){
  
  res_list <- list()
  res_list_SNP <- list()
  
  for(j in 1:length(harmonised_studies_sharedgenes[[i]])){

    dat <- harmonised_studies_sharedgenes[[i]][j]
    message("Performing MR for ", names(harmonised_studies_sharedgenes)[i],": ", names(harmonised_studies_sharedgenes[[i]])[j])

    res <- TwoSampleMR::mr(dat[[1]])
    res$pair <- names(harmonised_studies_sharedgenes)[i]
    res$study <- names(harmonised_studies_sharedgenes[[i]])[j]
    
    res_SNP <- TwoSampleMR::mr_singlesnp(dat[[1]]) |>
      left_join(dat[[1]][,c("SNP","pair","class","gene.exposure")])

    res_list <- c(res_list, list(res))
    res_list_SNP <- c(res_list_SNP, list(res_SNP))
  }
  res_join <- do.call("rbind", res_list)
  results_MR_sharedgenes <- c(results_MR_sharedgenes, list(res_join))
  
  res_join_SNP <- do.call("rbind", res_list_SNP)
  results_MR_sharedgenes_SNP <- c(results_MR_sharedgenes_SNP, list(res_join_SNP))
}

names(results_MR_sharedgenes) <- names(harmonised_studies_sharedgenes)
names(results_MR_sharedgenes_SNP) <- names(harmonised_studies_sharedgenes)

# Forest plot

res_sharedgenes_IVW <- do.call("rbind", results_MR_sharedgenes) |> filter(method == "Inverse variance weighted") |>
  mutate(instrument_type = instrument_type[study],
         instrument_class = factor(instrument_class[study], 
                                   levels = c(
                                     "genome:common",
                                     "exome:common","exome:lowfreq",
                                     "exome:rare","exome:rare (top)",
                                     "exome:ultrarare","exome:ultrarare (top)",
                                     "mask:synonymous","mask:missense|LC",
                                     "mask:pLOF|missense|LC","mask:pLOF",
                                     "genescore")))

p_ivw_shared <- ggplot(res_sharedgenes_IVW, aes(x = b, y = instrument_class, colour = instrument_type)) +
  facet_wrap(.~pair, nrow = 2, scale = "free_x") +
  geom_vline(xintercept = 0, colour = "grey20", lty = 2) +
  geom_point(aes(pch = pval<0.05, size = nsnp)) +
  geom_errorbar(aes(xmin = b-1.96*se, xmax = b+1.96*se), width = 0.1) +
  scale_colour_manual(values = c("orange","darkblue")) +
  scale_shape_manual(values = c(1,19)) +
  theme_bw() +
  labs(size = "n (instruments)", colour = "Type", x = "IVW", y = "")

gridExtra::grid.arrange(p_genes_shared, p_ivw_shared, nrow = 1, widths = c(0.4,0.6))

Comparison of Wald Ratio (subsetting to shared genes)

A comparison of Wald ratios retuned for common and rare instruments that are hitting the same genes

# lapply(results_MR_sharedgenes_SNP, function(x){range(x$b)})
# 
# plot_wald <- function(snp_results, set_1, set_2){
#   
#   axis_lim <- max(abs(range(snp_results$b))) + 1
# 
#   dat <- snp_results[,c("b","se","p","pair","class","gene.exposure")]
#   dat <- left_join(dat |> filter(class %in% set_1), dat |> filter(class %in% set_2), by = c("pair", "gene.exposure"), suffix = c(".1",".2"))
#   
#   ggplot(dat, aes(x = b.1, y = b.2, colour = gene.exposure)) +
#     geom_abline(slope = 1, intercept = 0, colour = "grey40") +
#     geom_errorbar(aes(xmin = b.1-1.96*se.1, xmax = b.1+1.96*se.1), width = 0.1) +
#     geom_errorbar(aes(ymin = b.2-1.96*se.2, ymax = b.2+1.96*se.2), width = 0.1) +
#     geom_point(size = 3) +
#     scale_colour_manual(name = "Gene/proximal gene", values = RColorBrewer::brewer.pal(name = "Paired", n = length(unique(dat$gene.exposure)))) +
#     scale_x_continuous(limits = c(-axis_lim, axis_lim)) +
#     scale_y_continuous(limits = c(-axis_lim, axis_lim)) +
#     theme_bw() +
#     theme(legend.position = "bottom",
#           plot.title = element_text(hjust = 0.5)) +
#     labs(x = paste0("Wald ratio (instrument class: ", set_1, ")"), 
#          y = paste0("Wald ratio (instrument class: ", set_2, ")"),
#          title = unique(dat$pair))
# }
# 
# lapply(results_MR_sharedgenes_SNP, plot_wald, set_1 = "exome:common", set_2 = "exome:rare")

Heterogeneity analysis

MR Egger intercept (horizontal pleiotropy)

# Perform MR on each exposure-outcome pair, across each of 12 instrument sets
res_egger <- list()

for(i in 1:length(harmonised_studies)){
  res_list <- list()
  
  for(j in 1:length(harmonised_studies[[i]])){
    dat <- harmonised_studies[[i]][j]
    
    if(nrow(dat[[1]]) == 1){
      res <- data.frame(
        id.exposure = NA,
        id.outcome = NA,
        outcome = NA,
        exposure = NA,
        egger_intercept = NA,
        se = NA,
        pval = NA
      )
    } else {
      res <- TwoSampleMR::mr_pleiotropy_test(dat[[1]])
    }
    
    res$pair <- names(harmonised_studies)[i]
    res$study <- names(harmonised_studies[[i]])[j]
    
    res_list <- c(res_list, list(res))
  }
  res_join <- do.call("rbind", res_list)
  res_egger <- c(res_egger, list(res_join))
}

res_egger <- do.call("rbind", res_egger) |>
  mutate(instrument_type = instrument_type[study],
         instrument_class = factor(instrument_class[study], 
                                   levels = c(
                                     "genome:common",
                                     "exome:common","exome:lowfreq",
                                     "exome:rare","exome:rare (top)",
                                     "exome:ultrarare","exome:ultrarare (top)",
                                     "mask:synonymous","mask:missense|LC",
                                     "mask:pLOF|missense|LC","mask:pLOF",
                                     "genescore")))

ggplot(res_egger, aes(x = egger_intercept, y = instrument_class, colour = instrument_type)) +
  facet_wrap(.~pair, nrow = 2, scale = "free_x") +
  geom_vline(xintercept = 0, colour = "grey20", lty = 2) +
  geom_point(aes(pch = pval<0.05), size = 3) +
  geom_errorbar(aes(xmin = egger_intercept-1.96*se, xmax = egger_intercept+1.96*se), width = 0.1) +
  scale_colour_manual(values = c("tomato","orange","darkblue")) +
  scale_shape_manual(values = c(1,19)) +
  theme_bw() +
  labs(x = "Egger intercept", colour = "Type", x = "IVW", y = "")